Sequencing of oligosaccharides

ABSTRACT

A process suitable for use in the sequencing of an oligosaccharide compound includes a first analysis of a primary oligosaccharide compound&#39;s monosaccharide composition, selection of sequencing agents to apply to the oligosaccharide compound, applying a selected sequencing agent to the oligosaccharide compound, and analyzing a released monosaccharide to select a second sequencing agent. The sequencing agent may be, for example, an enzyme. The oligosaccharide compound may be, for example, an oligosaccharide, or a product of an oligosaccharide, or a species having an oligosaccharide portion.

The present invention relates to the analysis of oligosaccharides andmore particularly to the form of analysis known as sequencing ofoligosaccharides.

According to one aspect of the present invention there is providedapparatus suitable for use in the sequencing of an oligosaccharide whichapparatus includes means for selecting a sequencing agent to be appliedto an oligosaccharide entity.

According to another aspect of the present invention there is provided aprocess suitable for use in the sequencing of an oligosaccharide whichprocess includes the use of a means for selecting a sequencing agent tobe applied to an oligosaccharide entity.

The oligosaccharide entity may be, for example, an oligosaccharide, or aproduct of an oligosaccharide, or a species having an oligosaccharideportion. A product of an oligosaccharide may be, for example, a productproduced by previously applying a sequencing agent to anoligosaccharide; by way of example, the product may itself be anoligosaccharide.

Oligosaccharides form a class of chemical compounds which are each madeup of a number of monosaccharide units linked together by glycosidicbonds. Important sources of naturally occurring oligosaccharides areglycoproteins in which saccharides are found linked to a peptide chaineither by an N-glycosidic bond or by an O-glycosidic bond; theseoligosaccharides may vary from a few monosaccharide units to highlybranched structures containing many (e.g. over 30) monosaccharide units.

The "sequencing" of an oligosaccharide involves deducing certaininformation concerning the structure of the oligosaccharide such as (i)the type of each monosaccharide unit in the oligosaccharide, (ii) theorder in which the monosaccharide units are arranged in theoligosaccharide, (iii) the position of linkages between each of themonosaccharide units (e.g. 1-3, 1-4, etc.), and hence any branchingpattern and/or (iv) the orientation of linkage between each of themonosaccharide units (i.e. whether a linkage is an α linkage or a βlinkage).

Where it is desired to obtain as much information as possible regardingthe structure of an oligosaccharide then "sequencing" of theoligosaccharide may be carried out to obtain as much information aspossible in relation to features (i) to (iv) inclusively immediatelyhereinbefore disclosed.

An agent which assists in obtaining information in relation to some orall of features (i) to (iv) inclusively on being applied to anoligosaccharide entity may be regarded as a "sequencing agent". By wayof example, a sequencing agent may be a physical agent or a chemicalagent. Examples of physical sequencing agents are proton n.m.r.,carbon-13 n.m.r. and mass spectrometry for molecular weightdeterminations.

Also, by way of example, a sequencing agent may be capable of causingcleavage of a chemical bond or capable of causing formation of achemical bond.

Where, for example, a sequencing agent is a chemical reagent (which maybe, for example, a chemical reagent or a biochemical reagent) thesequencing agent may be regarded as a sequencing reagent. Examples ofsequencing reagents are enzymes (such as exoglycosidases andendoglycosidases) and chemical reagents (e.g. a periodate) capable ofeffecting chemical cleavage of an oligosaccharide and/or a chemicalmodification of an oligosaccharide which assists in obtaininginformation regarding the structure of the oligosaccharide ashereinbefore disclosed.

As hereinbefore disclosed the oligosaccharide entity may be, forexample, an oligosaccharide, or a product of an oligosaccharide, or aspecies having an oligosaccharide portion.

Thus, it is to be understood that, by way of example, an oligosaccharideas such may be subjected to sequencing in accordance with the presentinvention; by way of further example, as an alternative, a product of anoligosaccharide may be subjected to sequencing in accordance with thepresent invention. (It will be appreciated that the product may itselfbe an oligosaccharide.)

Alternatively, for example, an oligosaccharide provided as anoligosaccharide portion of a species having an oligosaccharide portion(e.g. an oligosaccharide linked to a conjugate) may be subjected tosequencing in accordance with the present invention. Glycoproteins andglycolipids are examples of species having a portion comprising anoligosaccharide which may be subjected to sequencing in accordance withthe present invention such that oligosaccharide is subjected tosequencing.

Thus, by way of further example, an oligosaccharide may, if desired, besubjected to sequencing in accordance with the present invention whilststill attached to a conjugate thereof (e.g. a peptide chain) providedthat the conjugate does not interfere in the sequencing to anyunacceptable degree.

From the foregoing disclosure it will be appreciated that, by way ofexample, an oligosaccharide to be subjected to sequencing may beprovided in any suitable form and in any suitable manner.

Also, from the foregoing disclosure it will be appreciated that inaccordance with the present invention sequencing of an oligosaccharidemay include, for example, applying a sequencing agent to anoligosaccharide, or a product thereof, or a species having anoligosaccharide portion.

Examples of enzymes which may be used as sequencing reagents are givenin Table I.

In Table I there is presented a list of enzymes commonly used forcleaving monosaccharides from N-linked oligosaccharides and the rulesshowing which monosaccharides are cleaved by each of these enzymes andfrom which part of an oligosaccharide structure cleavage can beexpected.

A sequencing agent which is capable of bringing about a cleaving of aparticular linkage or linkages in an oligosaccharide may be an agentcapable of effecting a specific transformation on the oligosaccharide.

It will be appreciated that a sequencing agent may be chosen such thatthe reaction products obtained when it is applied to the oligosaccharideentity (e.g. contacted with the oligosaccharide entity in the case of achemical or biochemical reagent) will reveal the presence or absence ofa particular structural sub-unit (e.g. a monosaccharide unit) in theoligosaccharide entity.

                  TABLE I                                                         ______________________________________                                                      Monosaccharide                                                  Enzyme        Cleaved     Rules for Cleavage                                  ______________________________________                                        1)  Achatina fulica beta                                                                        Mannose     1-beta-4 to any site                                mannosidase                                                               2)  A. saitoi alpha                                                                             Mannose     1-alpha-2 to any site                               mannosidase                                                               3)  Jack bean alpha                                                                             Mannose     (i) mannose 1-alpha-2 to                            mannosidase               any site                                                                      (ii) cleaves the 1-alpha-                                                     3,6 mannoses if there is                                                      no bisect on the middle                                                       mannose                                         4)  Jack bean alpha                                                                             Mannose     Different from 3) in that                           mannosidase (under        it will not cleave the                              arm-specific              mannose 1-alpha-6 case                              conditions)               when restricted by a side                                                     arm longer than 1 unit                                                        out from the middle                                                           mannose                                         5)  Bovine testis beta                                                                          Galactose   1-beta-3,4 to any                                   galactosidase             non-branched site                               6)  Jack bean beta                                                                              Galactose   1-beta-4 to any                                     galactosidase             non-branched site                               7)  C. lampas beta                                                                              Xylose      1-beta-any to any site                              xylosidase                                                                8)  S. pneum beta N-acetyl    (i) 1-beta-3 to any non-                            N-acetyl      hexosamine  branched site                                       hexosaminidase            (ii) 1-beta-2 mannose                                                         1-alpha-3 or 6 to middle                                                      mannose provided the                                                          first mannose does not                                                        have a bond at atom 6,                                                        and also in the case of                                                       1-alpha-6, that the                                                           middle mannose is not                                                         bisected                                        9)  Jack bean beta                                                                              N-acetyl    1-beta-any to any site                              N-acetyl      hexosamine                                                      hexosaminidase                                                            10) Bovine epididymis                                                                           Fucose      1-alpha-3,4,6 to any site                           alpha fucosidase                                                          11) C. lampas alpha                                                                             Fucose      1-alpha-2,3,4,6 to any                              fucosidase                site                                            12) Coffee bean alpha                                                                           Galactose   1-alpha-3,6 to any                                  galactosidase             non-branched site                               13) Almond alpha  Fucose      1-alpha-3,4 to any site                             fucosidase                                                                ______________________________________                                    

Thus, in one embodiment of the present invention there is provided aprocess for the sequencing of an oligosaccharide which process includesapplying a sequencing agent to an oligosaccharide entity and analysingfor a component of the oligosaccharide entity, which component has beenreleased from the oligosaccharide entity by means of the sequencingagent.

It will be appreciated that in accordance with the immediately foregoingembodiment of the present invention it is not the oligosaccharide entitywhich is analysed after application of the sequencing agent to obtaininformation regarding the structure of the oligosaccharide entity.Rather, in accordance with the immediately foregoing embodiment of thepresent invention, it is a component which is released or cleaved froman oligosaccharide entity that is analysed for in order to facilitate"sequencing".

Thus, for example, when analysis is carried out, the detection of acomponent comprising a particular monosaccharide in the products of acleaving reaction may be used to confirm the presence of a particularlinkage and monosaccharide in the original oligosaccharide structure ofthe oligosaccharide entity.

In another embodiment of the present invention there is providedapparatus which includes means for selecting a sequencing agent to beapplied to an oligosaccharide entity and analysing means for analysingfor a component of the oligosaccharide entity, which component has beenreleased from the oligosaccharide entity by means of a sequencing agent.

A set of possible structures for an oligosaccharide (i.e. a set of"candidate" structures) may be prepared in any suitable way.

For example, a set of "candidate" structures may be drawn up fromliterature surveys.

By way of further example, a set of "candidate" structures may beprepared by considering possible permutations of putting togethermonosaccharide units.

Alternatively, by way of further example, a concept of structures andsub-structures may be used (as discussed further hereinafter) to preparea set of candidate structures for an unknown oligosaccharide.

It will also be appreciated that, for example, by sequentially applyingdifferent sequencing agents to an oligosaccharide entity and analysingthe products obtained by use of each sequencing agent it is possible toeliminate certain structures from consideration (i.e. eliminate certainstructures from a postulated set of possible "candidate" structures forthe oligosaccharide entity) and to confirm the presence of a certainstructure or certain structures thereby enabling information regardingthe structure of the oligosaccharide entity to be deduced.

Thus, for example, by sequentially applying different sequencing agentseither to a given oligosaccharide entity, comprising an oligosaccharide,or to a product thereof (being a product produced by previously applyinga sequencing agent to the oligosaccharide), or to a species having anoligosaccharide portion, and analysing the products obtained by use ofeach sequencing agent it is possible to eliminate certain structuresfrom consideration (i.e. eliminate certain structures from a postulatedset of possible "candidate" structures for the oligosaccharide) and toconfirm the presence of a certain structure or certain structuresthereby enabling information regarding the structure of theoligosaccharide (which may be, for example, an oligosaccharide as suchor an oligosaccharide portion of a species having an oligosaccharideportion) to be deduced.

The presence and linkage of a particular monosaccharide at an end of anoligosaccharide structure of an oligosaccharide entity may bedetermined, for example, by the ability of a given sequencing agent(e.g. a biochemical reagent such as an enzyme (e.g. an exoglycosidase))to cause cleavage of that linkage; thus, if cleavage occurs, thendetection of the particular monosaccharide in the reaction products ofthe cleaving reaction will confirm the presence of that linkage in theoriginal oligosaccharide structure. Thus, by sequentially using aplurality of different sequencing agents having known specific linkagecleaving capabilities it is possible to deduce increasing amounts ofinformation regarding the structure of the oligosaccharide entity underanalysis. It is to be understood that where no single sequencing agentcan be found which can distinguish between candidate oligosaccharidestructures then consideration may be given to possible combinations oftwo, three or more agents to be applied one after the other; at eachstage of consideration an agent which has no effect on any of a set ofcandidate structures may be eliminated. Consideration may also be givento using a combination of two or more agents simultaneously as this maylead to a reduction in the time required to carry out a sequencinganalysis.

Thus, an iterative process may be used whereby a cycle of analysis,application of a sequencing agent (or a combination of sequencingagents) and subsequent analysis is repeated until as much information aspossible has been obtained regarding the structure of an oligosaccharideentity with the agents available or the sample of oligosaccharide entityis exhausted.

From the foregoing it will be appreciated that in certain circumstancesa particular sequencing agent may be such that it does not react withthe oligosaccharide entity to give products thereby permitting the factthat it did not so react to allow deductions to be made regarding thestructure of the oligosaccharide entity.

The effectiveness of the sequencing of an oligosaccharide entity may beseen as depending upon the choice of sequencing agent to be applied atvarious stages in sequencing and the accuracy of interpretation of theresults of applying a given sequencing agent to an oligosaccharideentity.

To a large degree, a good choice of sequencing agent depends upon theskill of an experienced operator who has already made some intelligentguesses about the type of oligosaccharide structure being investigated.

A poor choice of sequencing agent may result in little or no additionalinformation being revealed by a particular application of a sequencingagent and thus lead to a waste of time and materials. Also there ispresent the danger that prejudices of an operator will mask ambiguitiesin the interpretation of results; for example, an operator may assign asingle structure which is consistent with experimental results, whereasin reality there may be more than one structure consistent with the sameexperimental results.

A further difficulty may arise in defining the point in sequencing atwhich no further information can be revealed by the use of availablesequencing agents.

For oligosaccharide entities (e.g. oligosaccharides) obtained fromglycoproteins the sequencing thereof may be assisted by a knowledge ofthe biosynthetic pathways involved in building up oligosaccharidestructures. Thus, for example, for N-linked oligosaccharides it is knownthat there is a characteristic core structure and that additionalmonosaccharides may only add on in certain well defined orders andbranching patterns.

This knowledge may be used to develop for oligosaccharides a concept ofstructures and sub-structures; this is discussed further hereinafterwith reference to FIGS. 1, 2, and 3 of the accompanying drawings.

For example, if an oligosaccharide entity to be subjected to sequencingin accordance with the present invention is an oligosaccharide which hasbeen obtained from a mixture of oligosaccharides released from aglycoprotein by the enzyme peptide-N-glycosidase F, it may be assumedthat the oligosaccharide is an N-glycan and that the structure thereofis likely to be a structure similar to those of FIG. 1, FIG. 2 or FIG. 3of the accompanying drawings or a sub-structure generated from thestructures similar to those of FIGS. 1, 2 and 3 of the accompanyingdrawings.

In one embodiment of an apparatus in accordance with the presentinvention an apparatus includes means for applying a sequencing agent toan oligosaccharide entity, analysing means for analysing productsobtained by applying the sequencing agent to an oligosaccharide entity,means for selecting a sequencing agent to be applied to anoligosaccharide entity, and means for feeding analysis results from theanalysing means to the means for selecting a sequencing agent to beapplied to an oligosaccharide entity.

In another embodiment of apparatus in accordance with the presentinvention an apparatus includes all of the features of the apparatusimmediately hereinbefore disclosed in the immediately precedingparagraph and additionally includes means for applying to anoligosaccharide entity, a sequencing agent as selected by the means forselecting a sequencing agent.

Apparatus in accordance with the present invention may also include ameans for carrying out a preliminary analysis of an oligosaccharideentity; it will be appreciated that this means optionally may be, or maybe part of, the analysing means for analysing products obtained byapplying the sequencing agent to an oligosaccharide entity. Thus, it isto be understood that, if desired, the analysing means may be onecapable of detecting monosaccharide units from an oligosaccharideentity.

The apparatus may also, optionally, include means for feeding theresults of a preliminary analysis to the means for selecting asequencing agent to be applied to an oligosaccharide entity.

The means for applying the sequencing agent to an oligosaccharide entitymay comprise, for example, a means for contacting together a sequencingagent and an oligosaccharide entity (e.g. a reaction vessel).

Also, the apparatus may include, for example, means for supplying asequencing agent to a means for contacting together a sequencing agentand an oligosaccharide entity.

The means for supplying a sequencing agent may be, for example, suchthat a plurality of sequencing agents may be supplied individually ormay be, for example, such that sequencing agents may be supplied in aselected sequence or combination.

A sequencing agent, or a combination of sequencing agents, may beapplied to an oligosaccharide entity in a suitable manner.

The sequencing agent or a combination of sequencing agents may beintroduced in a suitable solvent to a means for contacting together asequencing agent and an oligosaccharide entity.

Optionally, for example, an oligosaccharide entity, to be subjected tosequencing may be immobilised on a suitable support material.

Thus, for example, an apparatus in accordance with the present inventionmay include a support material upon which may be immobilised anoligosaccharide entity, to be subjected to sequencing.

Also, for example, a process in accordance with the present inventionmay include the step of immobilising upon a support material anoligosaccharide entity, to be subjected to sequencing.

The oligosaccharide entity may, for example, be immobilised on a supportmaterial by any suitable means. Thus, where, for example, theoligosaccharide entity is an oligosaccharide or a product thereofimmobilisation may be effected, for example, by means of chemicalattachment (e.g. covalent linkage) via a reducing terminus of anoligosaccharide.

An oligosaccharide entity comprising an oligosaccharide, or a productthereof, may be immobilised in accordance with the present invention forexample by direct covalent linkage with a support material, or by directnon-covalent (e.g. hydrophilic) linkage with a support material.

Alternatively, by way of example, an oligosaccharide entity comprising aspecies having an oligosaccharide portion may be immobilised on asupport material before being subjected to sequencing.

By way of example, where it is desired to immobilise an oligosaccharide,or a product thereof, whilst still attached to a conjugate the conjugatemay be linked to a support material (e.g. by covalent linkage ornon-covalent (e.g. hydrophilic) linkage) such that the oligosaccharide,or product thereof, is indirectly linked to the support material via theconjugate.

Where an oligosaccharide entity is immobilised on a support material theoligosaccharide entity, or any product thereof produced by applicationof the sequencing agent and retained on the support material, may bereadily separated from released products, such as species (with newreducing termini), generated by the application of a sequencing agent oragents. Thus, for example, the product of the oligosaccharide entity maybe retained on the support material and species with new reducingtermini may be removed by suitable washing.

An example of a support material for use in accordance with the presentinvention is a solid support material comprising 1,1'carbonyldiimidazoleactivated agarose.

Immobilisation of an oligosaccharide entity on a support material may beeffected by any suitable means. Thus, for example, where theoligosaccharide entity is an oligosaccharide, or a product of anoligosaccharide, immobilisation of an oligosaccharide, or a product ofan oligosaccharide, on a support material may be effected by thefollowing procedure:

unreduced oligosaccharide+2-amino pyridine/NaBH₃ CN→oligo-pyridylaminoderivative→conjugation.

By way of further example, where an oligosaccharide entity is anoligosaccharide or a product of an oligosaccharide, the oligosaccharideor product thereof may be immobilised on a support material by means ofthe following procedure:

unreduced oligosaccharide+dansyl hydrazine/TFA→oligo-dansyl hydrazinederivative,

oligo-dansyl hydrazine derivative+NaBH₄ /H₂ O→conjugation.

It is to be understood that attachment of an oligosaccharide entity, viaa reducing terminus, to a support material, is preferably independent ofany oligosaccharide structure present in the oligosaccharide entity; theattachment may not, for example, require a reducing terminusmonosaccharide to be retained in a ring-closed configuration.

Apparatus in accordance with the present invention may include, forexample, a filtration means (e.g. a filtration column involvingchromatographic separation or other chemical separation means) to permitremoval of excess sequencing agent or agents from a reaction mixture(e.g. formed in a reaction vessel) prior to using an analysing means.

An analysing means for use in accordance with the present invention mayinclude a detector capable of measuring the types and relative amountsof monosaccharides present in an oligosaccharide and/or monosaccharidesproduced by applying a sequencing agent to an oligosaccharide entity,(e.g. by bringing together a sequencing agent and an oligosaccharideentity). By way of example, the monosaccharides may be measured asmonosaccharides or as derivatised products thereof.

Apparatus in accordance with the present invention may also, forexample, include a flushing means whereby excess sequencing agent may beremoved from a means for contacting together a sequencing agent and anoligosaccharide entity (e.g. a reaction vessel) prior to theintroduction of a further sequencing agent.

A means for contacting together a sequencing agent and anoligosaccharide entity may be, for example, supplied with means formaintaining a controlled temperature.

It has been hereinbefore disclosed that an oligosaccharide entity to besubjected to analysis in accordance with the present invention may, forexample, be immobilised on a suitable support material. However, by wayof further example, an oligosaccharide entity may be subjected toanalysis in accordance with the present invention without suchimmobilisation; thus, for example, an oligosaccharide entity may haveapplied thereto a sequencing agent, or a sequence or a plurality ofsequencing agents, whilst in free solution.

In apparatus in accordance with the present invention the means forselecting a sequencing agent to be applied to an oligosaccharide entity,may be, for example, a unit capable of making logical choices (e.g. alogic unit).

Also, the unit may be such that it is capable of interpreting resultsgenerated by the analysing means and capable of selecting a sequencingagent (or a combination of sequencing agents) to be applied to anoligosaccharide entity. Thus, for example, the unit may be such as to becapable of requesting the application of a sequencing agent (or acombination of sequencing agents) to an oligosaccharide entity, theapplication of another sequencing agent (or combination of sequencingagents) to an oligosaccharide entity being a product of anoligosaccharide entity or the application of another or a furthersequencing agent (or combination of sequencing agents) to anoligosaccharide entity, or to an oligosaccharide entity being a productof an oligosaccharide entity.

It is to be understood that, by way of example, an apparatus inaccordance with the present invention may be such that, in operation, aunit as hereinbefore disclosed controls application of a sequencingagent or agents to an oligosaccharide entity to be analysed (inimmobilised form or otherwise), and interprets the output from adetector such as to provide a fully automated apparatus for theidentification and sequencing of an unknown oligosaccharide structure ofan oligosaccharide entity.

Thus, for example, an apparatus in accordance with the present inventionmay be such that, in operation, a unit as hereinbefore disclosedcontrols addition of a sequencing agent or agents to a reaction vessel,containing (in immobilised form or otherwise) an oligosaccharide entityto be analysed, and interprets the output from a detector such as toprovide a fully automated apparatus for the identification andsequencing of an unknown oligosaccharide structure of an oligosaccharideentity.

An apparatus in accordance with the present invention may be arranged,for example, to produce results of identification and sequencing of anoligosaccharide structure of an oligosaccharide entity in any suitablemanner.

In one embodiment of a process in accordance with the present inventiona process includes analysing an oligosaccharide entity, applying asequencing agent to an oligosaccharide entity, analysing productsobtained by applying the sequencing agent to an oligosaccharide entityand feeding analysis results thus obtained to a means for selecting asequencing agent to be contacted with an oligosaccharide entity.

In another embodiment of a process in accordance with the presentinvention a process includes all of the steps of the processhereinbefore disclosed in the immediately preceding paragraph and alsoadditionally includes the step of applying a sequencing agent, asselected by the means for selecting a sequencing agent, to anoligosaccharide entity.

A process in accordance with the present invention may also include thestep of carrying out a preliminary analysis of the oligosaccharideentity of unknown structure prior to application of a sequencing agent.

The results of such an analysis may be fed to the means for determininga sequencing reagent.

It will be appreciated that a preliminary compositional analysis mayenable the number of candidate oligosaccharide structures, that need tobe considered during subsequent structural analysis, to be reduced;thus, such a preliminary structural analysis may enable the number ofsequencing agent applications to be reduced.

The analysis of the oligosaccharide entity (e.g. a preliminary analysisor any subsequent analysis) may be effected in any suitable way. Thus,for example, the type and number of each monosaccharide in theoligosaccharide entity may be analysed (i.e. a compositional analysismay be effected) by any suitable method. For example, completedegradation of an oligosaccharide structure of an oligosaccharide entityinto its monosaccharide components may be effected by treatment withsuitable reagents (e.g. a mixture of digesting reagents such asexoglycosidases) and the resulting reaction mixture analysed using asuitable monosaccharide detection method such as those hereinafterdisclosed. Alternatively, by way of further example, an oligosaccharideentity may be subjected to methanolysis, N-acetylation (if required) andsilylation and the resulting substances subjected to gaschromatography/mass spectrometry.

It will be appreciated that, by way of further example, informationregarding an oligosaccharide structure of an oligosaccharide entity mayalso be obtained by observing its retention time on a chromatographiccolumn; by way of example the chromatographic column may be such thatthe retention time of an oligosaccharide entity is expressed in glucoseunits.

Monosaccharide detection may be effected in any suitable manner,examples of which are the following HPLC-based methods: (i) use of an SP1010 reverse phase column, (ii) HPAE with PAD using a Dionex instrumentand (iii) capillary electrophoresis.

It is to be understood that the present invention offers the possibilityof optimising the use of a sequencing agent or sequencing agents,interpreting the results of an analysis of a reaction between anoligosaccharide entity and a sequencing agent unambiguously, anddetermining the point where no further sequencing with an availablesequencing agent or agents is possible.

It will be appreciated that in accordance with the present invention itis, for example, possible to achieve a "loop" in which informationobtained from a means for applying a sequencing agent to anoligosaccharide entity (or a product thereof produced by the effect of asequencing agent upon an oligosaccharide entity) as detected by adetector is fed to a means for selecting a sequencing agent to beapplied to an oligosaccharide entity (or a product thereof, produced bythe effect of a sequencing agent upon the oligosaccharide entity) andthe selection of sequencing agent to be next applied, as made by themeans for selecting a sequencing agent, is fed to the means for applyinga sequencing agent such that an iterative cycle may be established. Byway of example, such an iterative cycle may be carried out in a fullyautomated apparatus such that a sample oligosaccharide entity may beintroduced into a means for applying a sequencing agent and theapparatus allowed to function until sequencing of an oligosaccharidestructure of an oligosaccharide entity ceases, or a desired degree ofsequencing has taken place.

Thus, in one embodiment of the present invention there is provided anapparatus comprising a means for applying a sequencing agent to anoligosaccharide entity, means for detecting products obtained byapplying the sequencing agent to an oligosaccharide entity, and meansfor selecting a sequencing reagent to be applied to an oligosaccharideentity, the arrangement being such that, in operation, an iterativecycle can be achieved whereby the results of applying a sequencing agentselected by the means for selecting a sequencing agent are fed back, viathe means for detecting products, to the means for selecting asequencing agent whereby the means for selecting a sequencing agent maycause a further sequencing agent to be applied to an oligosaccharideentity, or product thereof.

BRIEF DESCRIPTION OF DRAWINGS

The present invention will now be further described, by way of exampleonly, with reference to the accompanying drawings and with reference tothe Examples.

In the accompanying drawings:

FIG. 1 shows a structure for N-linked oligosaccharides of high mannosetypes (Man 9);

FIG. 2 shows a structure for N-linked oligosaccharides of hybrid types(Hy 2);

FIG. 3 shows a structure for N-linked oligosaccharides ofmulti-antennary types (Hex 2);

FIG. 4 shows a diagrammatic representation of an apparatus in accordancewith the present invention;

FIG. 5 shows a diagrammatic representation of another apparatus inaccordance with the present invention;

FIG. 6 shows a diagrammatic representation of an apparatus for use inaccordance with the present invention;

FIG. 7 shows a diagrammatic representation of further apparatus for usein accordance with the present invention;

FIG. 8 shows a total ion current chromatogram of TMS-methyl glycosidesof standard monosaccharides;

FIGS. 9 to 27 show Gas Chromatography-Mass Spectra for TMS-methylglycosides of standard monosaccharides. The relevant monosaccharide isindicated in the top right-hand corner of each Figure; it will beappreciated that M/Z in the Figures indicates mass/charge ratio;

FIG. 28 shows a structure of an oligosaccharide entity to whichreference is made in Example 1;

FIG. 29 shows a total ion current chromatogram of TMS-methyl glycosidesobtained as disclosed in relation to Example 1(a);

FIGS. 30 to 33 show Gas Chromatography-Mass Spectra for TMS-methylglycosides obtained as disclosed in relation to Example 1(a);

FIG. 34 shows a total ion current chromatogram of TMS-methyl glycosidesobtained as disclosed in relation to Example 1(b);

FIGS. 35 to 37 show Gas Chromatography-Mass Spectra for TMS-methylglycosides obtained as disclosed in relation to Example 1(b);

FIG. 38 shows a total ion current chromatogram of TMS-methyl glycosidesobtained as disclosed in relation to Example 1(c);

FIGS. 39 to 41 show Gas Chromatography-Mass Spectra of TMS-methylglycosides obtained as disclosed in relation to Example 1(c);

FIG. 42 shows a structure of an oligosaccharide entity to whichreference is made in Example 2;

FIG. 43 shows a structure of an oligosaccharide entity to whichreference is made in Example 3; and

FIG. 44 shows a structure of an oligosaccharide entity to whichreference is made in Example 4.

In FIGS. 1, 2 and 3 of the accompanying drawings and elsewhere in thisSpecification the abbreviations Man, Fuc, Gal and Glcnac mean,respectively, D-mannose, L-fucose, D-galactose andN-acetyl-D-glucosamine.

Referring now to FIG. 1 of the accompanying drawings there is shown astructure for N-linked oligosaccharides of high mannose types.

It will be appreciated that the structure shows a number of mannose andN-acetyl glucosamine monosaccharide units, linked by a variety oflinkages; it will also be appreciated that the N-acetyl glucosamine unitto the extreme right of the Figure may be identified as the reducingterminus of the structure. The concept of structures and sub-structureswas hereinbefore disclosed and it may now be stated that it may beassumed that an oligosaccharide, for which a particular structure isrelevant, is either the structure itself or is a member of a set ofsub-structures of the structure, which sub-structures may be generatedby performing a specific transformation on the structure. This leads toa possibility that successive sequencing of an oligosaccharide structureof an oligosaccharide entity will eliminate more and more candidatestructures from a set of structures until no further information can beobtained.

Thus, an unknown oligosaccharide structure of an oligosaccharide entitymay be identified as one of the structures remaining.

In the case of the structure shown in FIG. 1 (and in FIGS. 2 and 3) thetransformations used to generate sub-structures are successive deletionsof terminal monosaccharides in all possible ways; this forms all uniquesub-structures having the same root as the structure where thecombination of the monosaccharides existing in the sub-structure followsthat of the structure.

Referring now to FIG. 2 of the accompanying drawings there is shown astructure for N-linked oligosaccharides of hybrid types (Hy 2).

The disclosure regarding structure and sub-structures hereinbefore givenin relation to FIG. 1 applies mutatis mutandis in relation to FIG. 2.

Referring now to FIG. 3 of the accompanying drawings there is shown astructure for N-linked oligosaccharides of multi-antennary types (Hex2).

The disclosure regarding structure and substructures hereinbefore givenin relation to FIG. 1 applies mutatis mutandis in relation to FIG. 3.

Referring now to FIG. 4 of the accompanying drawings there is shown adiagrammatic representation of an apparatus in accordance with thepresent invention said apparatus having a means 1 for applying asequencing agent to an oligosaccharide entity, analysing means 2 foranalysing products obtained by applying the sequencing agent to anoligosaccharide entity, and means 3 for selecting a sequencing agent tobe applied to an oligosaccharide entity.

In operation an oligosaccharide entity (e.g. an oligosaccharide or aproduct thereof, or a species having an oligosaccharide portion), isintroduced into the means 1 and a first selected sequencing agent, or afirst selected combination of sequencing agents, is applied. Thereaction products thus obtained are analysed by means 2 and the resultsof the analysis passed to the means 3 via a link identified by route 4.The output of means 3 is fed back to means 1 via a link identified asroute 5 whereby a second selected sequencing agent, or a second selectedcombination of sequencing agents, is applied to the oligosaccharideentity, or a product thereof, produced by the effect of the firstselected sequencing agent, or the first selected combination ofsequencing agents, upon the oligosaccharide entity.

By way of example, a third sequencing agent, or a third combination ofsequencing agents, may then be applied on the basis of results obtainedby applying the second sequencing agent, or second combination ofsequencing agents, and so on.

Thus, it is possible to achieve a "loop" in which information obtainedby applying a sequencing agent, or a combination of sequencing agents,to an oligosaccharide entity, in means 1, as detected by means 2, is fedto means 3 and the selection of sequencing agent, or agents, to be nextapplied, as made by means 3, is fed to means 1 such that an iterativecycle may be established. It will be appreciated that, for example, acycle of applying a sequencing agent, analysis, and application of afurther sequencing agent may be repeated until as many sequencing agentsas desired have been used. Thus, for example, apparatus as hereinbeforedescribed with reference to FIG. 4 of the accompanying drawings may bearranged to be fully automated such that a sample oligosaccharide entitymay be introduced into means 1 and the apparatus allowed to functionuntil sequencing of an oligosaccharide structure of an oligosaccharideentity ceases, or a desired degree of sequencing has taken place.

The link identified as 6 indicates that means 1 and 2 may be incommunication in some suitable manner. Thus, for example, where means 2is capable of analysis by direct optical means the link 6 may beoptical. By way of further example, where means 2 is capable ofanalysing samples taken from means 1 the link 6 may be a means fortransferring a sample from means 1 to means 2.

Referring now to FIG. 5 of the accompanying drawings there is shown adiagrammatic representation of another apparatus in accordance with thepresent invention said apparatus having a unit 10 which has a reactionunit 11, a detector means 12 (which includes a capillary electrophoresisapparatus) and a means 13 for selecting a sequencing agent to be appliedto an oligosaccharide entity; said means 13 includes a logic unit.

In operation an oligosaccharide entity is introduced into the reactionunit 11 and a first selected sequencing agent, or a first selectedcombination of sequencing agents, is introduced. The reaction productsthus obtained are detected by the detector means 12 and the output ofthe detector means 12 is passed to the means 13 by a link indicated as14. The output of means 13 is fed back to reaction unit 11, by a linkindicated as 15, whereby a second selected sequencing agent, or a secondselected combination of sequencing agents, is applied to theoligosaccharide entity, or a product thereof produced by the effect ofthe first selected sequencing agent, or the first selected combinationof sequencing agents, upon the oligosaccharide entity.

By way of example, a third sequencing agent, or a third combination ofsequencing agents, may be applied on the basis of results obtained byapplying the second sequencing agent, or second combination ofsequencing agents, and so on.

Thus, it is possible to achieve a "loop" in which information obtainedby applying a sequencing agent, or a combination of sequencing agents,to an oligosaccharide entity, in reaction unit 11, as detected bydetector means 12, is fed to means 13 and the selection of sequencingagent, or sequencing agents, to be next applied, as made by means 13, isfed to reaction unit 11 such that an interactive cycle may beestablished. It will be appreciated that, for example, a cycle ofapplying a sequencing agent, analysis, and application of a furthersequencing agent may be repeated until as many sequencing agents asdesired have been used. Thus, for example, apparatus as hereinbeforedescribed with reference to FIG. 5 of the accompanying drawings may bearranged to be fully automated such that a sample oligosaccharide entitymay be introduced into reaction unit 11 and the apparatus allowed tofunction until sequencing of an oligosaccharide structure of anoligosaccharide entity ceases, or a desired degree of sequencing hastaken place.

Referring now to FIG. 6 of the accompanying drawings there is shown adiagrammatic representation of apparatus for use in accordance with thepresent invention which apparatus has a reaction vessel 20, a heater 21for heating the reaction vessel 20, a supply line 22, a supply line 23,a buffer reagent storage means 24, a sequencing agent storage means 25(which may be provided with a cooling means (not shown)), a separationmeans 26 and a collection vessel 27.

In operation an oligosaccharide entity to be subjected to sequencing maybe attached to a support material to form a conjugated material and theconjugated material located in the reaction vessel 20. By application ofnitrogen gas through supply lines 22 and 23 and, as appropriate, the useof valves (not shown in this diagrammatic representation), a bufferreagent or reagents may be moved from the buffer reagent storage means24, (via line 28, and supply line 23 and line 30) to the reaction vessel20, and a sequencing agent, or agents, may be moved from the sequencingagent storage means 25 (via line 29, a supply line 23 and line 30) tothe reaction vessel 20.

Heater 21 may be used to maintain the temperature of the reaction vessel20 at a selected temperature.

Thus, a sequencing agent, or agents, (together with a buffer reagent, orreagents, as desired) may be applied to the conjugated material in thereaction vessel 20.

By use of nitrogen gas and, as appropriate, the use of valves (not shownin this diagrammatic representation), reaction products formed in thereaction vessel 20 may be removed from the reaction vessel 20 (via line30 and supply line 23), passed through the separation means 26, whichmay contain a suitable substance for effecting the removal of unwantedmaterial, and collected as a sample, via line 31, in collection vessel27.

The sample in collection vessel 27 may be subjected to any suitableanalysis (e.g. by use of a capillary electrophoresis apparatus (notshown)) and the results of analysis supplied to a means (not shown) forselecting a sequencing agent to be applied to an oligosaccharide entitysuch that a further sequencing agent, or further sequencing agents, maybe selected and supplied to the reaction vessel 20 from the sequencingagent storage means 25 (together with buffer reagent from the bufferreagent storage means 24 as desired). In this way a "loop" may beestablished which enables an iterative cycle to be effected in whichsequencing may be carried out.

If desired, the line 31 may be arranged to supply a sample directly toan analysing means (not shown) (e.g. a means which includes a capillaryelectrophoresis apparatus) rather than to collection vessel 27.

Waste lines 32, 33 and 34 may be provided, as necessary, for thedischarge of unwanted materials from the apparatus.

Line 35 is provided so as to permit reaction vessel 20 to be connected(via valves (not shown) as appropriate) with supply line 22 or wasteline 32 as may be desired.

Referring now to FIG. 7 of the accompanying drawings there is shown adiagrammatic representation of further apparatus for use in accordancewith the present invention which apparatus has an oligosaccharide entitystorage vessel 40, a reaction vessel 41 (which may be provided, forexample, with a heater (not shown)), a supply line 42, a buffer reagentstorage means 43, a sequencing agent storage means 44 (which may beprovided with a cooling means (not shown)), a separation means 45 and acollection vessel 46.

In operation a supply of solution containing an oligosaccharide entityto be subjected to sequencing in accordance with the present inventionis placed in the oligosaccharide entity storage vessel 40. Subsequently,by the application of nitrogen gas through supply line 42 and, asappropriate, the use of valves (not shown in this diagrammaticrepresentation) a sample of the oligosaccharide entity may be moved (vialine 47 and supply line 42 and line 48) from the oligosaccharide storagevessel 40 to the reaction vessel 41 and, also, buffer reagent, orreagents, may be moved from the buffer reagent storage means 43 (vialine 49, supply line 42 and line 48) to the reaction vessel 41, and,further, a sequencing reagent, or reagents, may be moved from thesequencing reagent storage means 44 (via line 50, supply line 42 andline 48) to the reaction vessel 41.

Thus, a sequencing agent, or agents, (together with a buffer reagent, orreagents, as desired) may be mixed with a sample of oligosaccharideentity taken from the supply of oligosaccharide entity.

By use of nitrogen gas and, as appropriate, the use of valves (not shownin this diagrammatic representation), reaction products formed in thereaction vessel 41 may be moved (via line 48 and supply line 42), passedthrough the separation means 45, which may contain a suitable substancefor effecting the removal of unwanted material, and collected as asample, via line 51, in collection vessel 46.

The sample in collection vessel 46 may be subjected to any suitableanalysis (e.g. by use of a capillary electrophoresis apparatus (notshown) and the results of analysis supplied to a means (not shown) forselecting a sequencing agent to be applied to an oligosaccharide entitysuch that a further sequencing agent, or further sequencing agents, maybe selected and supplied (from the sequencing agent storage means 44) toa fresh sample of oligosaccharide entity (from the oligosaccharideentity storage vessel 40) in reaction vessel 41 (together with bufferreagent from the buffer reagent storage means 43 as desired). In thisway a "loop" may be established which enables an iterative cycle to beeffected in which sequencing may be carried out.

If desired, the line 46 may be arranged to supply a sample directly toan analysing means (not shown) (e.g. a means which includes a capillaryelectrophoresis apparatus) rather than to collection vessel 46.

Waste line 52 may be provided, as necessary, for the discharge ofunwanted materials from the apparatus.

EXAMPLE 1

In this Example an oligosaccharide entity comprising an oligosaccharideof the structure given in FIG. 28 of the accompanying drawings was usedto demonstrate use of the present invention.

The oligosaccharide was confirmed as having a purity of >95% by 500 MH₃¹ H-NMR (1-dimensional) and high performance anion-exchangechromatography.

The oligosaccharide was immobilised by being conjugated to a supportmaterial comprising, 1,1' carbonyl diimidazole-activated agarose usingreductive amination as follows:

1 mg of the oligosaccharide was heated with 80 μl of a reagent preparedby dissolving 100 mg 2-amino pyridine in 65 μl of concentratedhydrochloric acid at 90° C. for 12 minutes. Subsequently, 8 μl of adimethyl sulphoxide solution of sodium cyanoborohydride at concentration1.66 gm/ml was added and the resulting mixture heated at 90° C. for afurther 90 minutes. After cooling, the mixture was diluted with 0.5 mln-butanol:ethanol (4:1) then applied to a column of cellulose (4 ml) andeluted with 20 ml n-butanol:ethanol:water [4:4:1], followed by methanol(3 ml) then water (5 ml). The methanol and water fractions were combinedand concentrated by rotary-evaporation to 0.2 ml. A slurry ofdiimidazolecarbonyl activated agarose in acetone (0.5 ml) was added andthe resulting mixture stirred at room temperature for 24 hours.

The combination of oligosaccharide conjugated to the support material(which combination will be referred to as "conjugated material" in thisExample) was separated from reaction mixture and any unconjugatedsubstances by rinsing in 0.1M sodium chloride followed by centrifugation(1000 g for 1 minute), the liquid supernatant being discarded; therinsing, centrifugation and discarding of liquid was repeated fivetimes.

The immobilised oligosaccharide was subjected to sequencing byincubating conjugated material with exoglycosidases and identifying andquantifying any released monosaccharides as follows:

A preliminary analysis of the oligosaccharide was carried out and thefollowing monosaccharide units were identified: Man (3 units), Gal (2units) and Glcnac (4 units).

The results of the preliminary analysis were used, in conjunction with ameans for selecting a sequencing agent to be applied to anoligosaccharide entity (said means including a logic unit), to identifycandidate structures and to select a sequencing agent to be applied tothe conjugated material.

Thus:

(a) to conjugated material in a plastic tube was added 100 μl of asolution consisting of 0.1M sodium citrate/phosphate, pH 3.5 containing1.0 unit of purified β-D-galactosidase enzyme (obtained from jack bean)to form a mixture. The tube was capped and the mixture incubated at 37°C. for 6 hours. Conjugated material was separated by rinsing in 0.1Msodium chloride followed by centrifugation (1000 g for 1 minute). Liquidsupernatant was separated from conjugated material and collected. Thiswas repeated and all liquid supernatant was pooled and desalted bypassage through an ion-exchange column consisting of 0.5 ml Dowex AG50X12(H⁺) resin below 0.5 ml Dowex AG3X 4A(OH⁻) resin. (Both resins werepurchased from Bio RAD.) Eluent from the column was collected,rotary-evaporated to dryness and converted to the 1-O-methyltrimethylsilyl glycoside exactly according to the standard procedure ofChaplin (Analytical Biochemistry 123 p.336 (1982)). The resulting1-O-methyl trimethylsilyl glycoside was quantitated by GC-MS andidentified by reference to known standard compounds based on retentiontime during GC and mass spectrum. The total ion current chromatogram ofstandard monosaccharides is shown in FIG. 8 of the accompanying drawingsand mass spectra of standard monosaccharides are shown in FIGS. 9 to 27of the accompanying drawings. The total ion current chromatogram andmass spectra for the liquid supernatant recovered after incubating theconjugated material with the β-D-galactosidase are shown respectively inFIGS. 29 and 30 to 33 of the accompanying drawings. From thisinformation it can be concluded that the action of the β-D-galactosidaseled to the separation from the conjugated material of 511 nanomoles ofgalactose and of no other monosaccharide;

(b) the information obtained in (a) above was used in conjunction with ameans for selecting a sequencing agent to be applied to anoligosaccharide entity (said means including a logic unit), to select afurther sequencing agent to be applied to the conjugated materialobtained after treatment as disclosed in (a) above.

Thus to conjugated material (as obtained after treatment as disclosed in(a) above) in a plastic tube was added 100 μl of a solution consistingof 0.1M sodium cacodylate, pH 6.0 containing 48 microunits of purifiedβ-N-acetyl-D-hexosaminidase enzyme (obtained from Streptococcuspneumoniae) to form a mixture. The tube was capped and the mixtureincubated at 37° C. for 6 hours. Conjugated material was separated byrinsing in 0.1M sodium chloride followed by centrifugation (1000 g for 1minute). Liquid supernatant was separated from conjugated material andcollected. This was repeated and all liquid supernatant was pooled anddesalted by passage through an ion-exchange column consisting of 0.5 mlDowex AG50X 12(H⁺) resin below 0.5 ml Dowex AG3X 4A(OH⁻) resin. (Bothresins were purchased from Bio RAD.) Eluent from the column wascollected, rotary-evaporated to dryness and converted to the 1-O-methyltrimethylsilyl glycoside exactly according to the standard procedure ofChaplin (Analytical Biochemistry 123 p.336(1982)). The resulting1-O-methyl trimethylsilyl glycoside was quantitated by GC-MS andidentified by reference to known standard compounds based on retentiontime during GC and mass spectrum. The total ion current chromatogram ofstandard monosaccharides is shown in FIG. 8 of the accompanying drawingsand mass spectra of standard monosaccharides are shown in FIGS. 9 to 27of the accompanying drawings. The total ion current chromatogram andmass spectra for the liquid supernatant recovered after incubating theconjugated material with the β-N-acetyl-D-hexosaminidase are shownrespectively in FIG. 34 and FIGS. 35 to 37 of the accompanying drawings.From this information it can be concluded that the action of theβ-N-acetyl-D-hexosaminidase led to the separation from the conjugatedmaterial of 487 nanomoles of N-acetylglucosamine and of no othermonosaccharide;

(c) the information obtained in (b) above was used in conjunction with ameans for selecting a sequencing agent to be applied to anoligosaccharide entity (said means including a logic unit), to select afurther sequencing agent to be applied to the conjugated materialobtained after treatment as disclosed in (b) above.

Thus, to conjugated material (as obtained after treatment as disclosedin (b) above) in a plastic tube was added 100 μl of a solutionconsisting of 0.1M sodium acetate/0.01M zinc acetate, pH 5.0 containing6 units of the purified α-D-mannosidase enzyme (obtained from jack bean)to form a mixture. The tube was capped and the mixture incubated at 37°C. for 6 hours. Conjugated material was separated by rinsing in 0.1Msodium chloride followed by centrifugation (1000 g for 1 minute). Theliquid supernatant was separated from conjugated material and collected.This was repeated and all liquid supernatant was pooled and desalted bypassage through an ion-exchange column consisting of 0.5 ml Dowex AG50X12(H⁺) resin below 0.5 ml Dowex AG3X 4A(OH⁻) resin. (Both resins werepurchased from Bio RAD.) Eluent from the column was collected,rotary-evaporated to dryness and converted to the 1-O-methyltrimethylsilyl glycoside exactly according to the standard procedure ofChaplin (Analytical Biochemistry 123 p.336 (1982)). The resulting1-O-methyl trimethylsilyl glycoside was quantitated by GC-MS andidentified by reference to known standard compounds based on retentiontime during GC and mass spectrum. The total ion current chromatogram ofstandard monosaccharides is shown in FIG. 8 of the accompanying drawingsand mass spectra of standard monosaccharides are shown in FIGS. 9 to 27of the accompanying drawings. The total ion current chromatogram andmass spectra for the liquid supernatant recovered after incubating theconjugated material with the α-D-mannosidase are shown respectively inFIG. 38 and FIGS. 39 to 41 of the accompanying drawings. From thisinformation it can be concluded that the action of the α-D-mannosidaseled to the separation from the conjugated material of 527 nanomoles ofmannose and of no other monosaccharide;

(d) the information obtained in (c) above was used, in conjunction witha means for selecting a sequencing agent to be applied to anoligosaccharide entity (said means including a logic unit), to select afurther sequencing agent to be applied to the conjugated materialobtained after treatment as disclosed in (c) above.

Thus, to conjugated material (as obtained after treatment as disclosedin (c) above) in a plastic tube was added 100 μl of a solutionconsisting of 0.1M sodium acetate, pH 4.0 containing 0.3 units of thepurified β-D-mannosidase enzyme (obtained from Helix pomatia) to form amixture. The tube was capped and the mixture incubated at 37° C. for 6hours. Conjugated material was separated by rinsing in 0.1M sodiumchloride followed by centrifugation (1000 g for 1 minute). The liquidsupernatant was separated from conjugated material and collected. Thiswas repeated and all liquid supernatant was pooled and desalted bypassage through an ion-exchange column consisting of 0.5 ml Dowex AG50X12(H⁺) resin below 0.5 ml Dowex AG3X 4A(OH⁻) resin. (Both resins werepurchased from Bio RAD.) Eluent from the column was collected,rotary-evaporated to dryness and converted to the 1-O-methyltrimethylsilyl glycoside exactly according to the standard procedures ofChaplin (Analytical Biochemistry 123 p.336 (1982)). The resulting1-O-methyl trimethylsilyl glycoside was quantitated by GC-MS andidentified by reference to known standard compounds based on retentiontime during GC and mass spectrum. The total ion current chromatogram ofstandard monosaccharides is shown in FIG. 8 of the accompanying drawingsand mass spectra of standard monosaccharide are shown in FIGS. 9 to 27of the accompanying drawings. The total ion current chromatogram andmass spectra for the liquid supernatant recovered after incubating theconjugated material with the β-D-mannosidase were essentially the sameas those shown, respectively, in FIGS. 38 and FIGS. 39 to 41 of theaccompanying drawings. From this information it can be concluded thatthe action of the β-D-mannosidase led to the separation from theconjugated material of 271 nanomoles of mannose and of no othermonosaccharide;

(e) the information obtained in (d) above was used, in conjunction witha means for selecting a sequencing agent to be applied to anoligosaccharide entity (said means including a logic unit) to select afurther sequencing agent to be applied to the conjugated materialsobtained after treatment as disclosed in (d) above.

Thus, to conjugated material (as obtained after treatment as disclosedin (d) above) in a plastic tube was added 100 μl of a solutionconsisting of 0.1M sodium citrate/phosphate, pH 4.5 containing 2.5 unitsof the purified β-N-acetyl-D-hexosaminidase (obtained from jack bean) toform a mixture. The tube was capped and the mixture incubated at 37° C.for 6 hours. Conjugated material was separated by rinsing in 0.1M sodiumchloride followed by centrifugation (1000 g for 1 minute). The liquidsupernatant was separated from conjugated material and collected. Thiswas repeated and all liquid supernatant was pooled and desalted bypassage through an ion-exchange column consisting of 0.5 ml Dowex AG50X12(H⁺) resin below 0.5 ml Dowex AG3X 4A(OH⁻) resin. (Both resins werepurchased from Bio RAD.) Eluent from the column was collected,rotary-evaporated to dryness and converted to the 1-O-methyltrimethylsilyl glycoside exactly according to the standard procedure ofChaplin (Analytical Biochemistry 123 p.336 (1982)). The resulting1-O-methyl trimethylsilyl glycoside was quantitated by GC-MS andidentified by reference to known standard compounds based on retentiontime during GC and mass spectrum. The total ion current chromatogram ofstandard monosaccharides is shown in FIG. 8 of the accompanying drawingsand mass spectra of standard monosaccharides are shown in FIGS. 9 to 27of the accompanying drawings. The total ion current chromatogram andmass spectra for the liquid supernatant recovered after incubating theconjugated material with β-N-acetyl-D-hexosaminidase were essentiallythe same as those shown, respectively, in FIG. 34 and FIGS. 35 to 37 ofthe accompanying drawings. From this information it can be concludedthat the action of the β-N-acetyl-D-hexosaminidase led to the separationfrom the conjugated material of 267 nanomoles of N-acetylglucosamine andof no other monosaccharide.

From the information obtained as above disclosed in this Example and thewell known specificities of the exoglycosidases employed, it is clearthat monosaccharides were released from the conjugated material in theorder and ratio stated below:

D-galactose β1→4: 2 residues

N-acetyl-D-glucosamine β1→2: 2 residues

Mannose α1→6,3: 2 residues

Mannose β1→4: 1 residue

N-acetyl-D-glucosamine β1→4: 1 residue

The monosaccharide attaching the starting oligosaccharide to the resinis not susceptible to an exoglycosidase enzyme, since it is not attachedby an O-glycosidic linkage. This terminal N-acetyl glucosamine (Glcnac)is therefore understood to exist. From this information, the sequence ofthe initial oligosaccharide can clearly only be that as shown in FIG. 28of the accompanying drawings.

This sequence is consistent with NMR studies of a solution form of theoligosaccharide.

EXAMPLE 2

In this Example an oligosaccharide entity comprising an oligosaccharideof the structure given in FIG. 42 of the accompanying drawings was usedto demonstrate use of the present invention.

A preliminary analysis of the oligosaccharide was carried out and thefollowing monosaccharide units were identified: Gal (2 units), Glcnac (5units), Man (3 units), Fuc (1 unit).

The oligosaccharide (0.6 mg) was attached to a support material asdisclosed in relation to Example 1 to give a conjugated material.

The results of the preliminary analysis were used, in conjunction with ameans for selecting a sequencing agent to be applied to anoligosaccharide entity (said means including a logic unit), to identifycandidate structures and to select a sequencing agent to be applied tothe conjugated material.

The conjugated material was subjected to successive treatments withvarious sequencing agents (in this Example exoglycosidase enzymes), thechoice of each successive sequencing agent being based upon the resultsof analysis and use of a means for selecting a sequencing agent to beapplied to an oligosaccharide entity (said means including a logicunit); the procedures used in this Example were substantially similar tothose disclosed in relation to Example 1.

The order in which the various sequencing agents (exoglycosidases inthis Example) were applied and the results are set out in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Order of Use      Exoglycosidase Reaction                                                                            Nanomoles of                           of Exoglyco-      Condition (all in 100 μl                                                               Monosaccharide                                                                         Monosaccharide                         sidase Exoglycosidase Used                                                                      reaction volume)                                                                          Detected Detected                               __________________________________________________________________________    1      β-D-galactosidase                                                                   1 unit in 0.1M sodium                                                                     Galactose                                                                              374                                           (jack bean)                                                                              citrate/phosphate,                                                            pH 3.5                                                      2      β-N-acetyl-D-                                                                       48 μunits in 0.1M                                                                      N-acetyl 179                                           hexosaminidase                                                                           sodium cacodylate,                                                                        glucosamine                                            (Streptococcus                                                                           pH 6.0                                                             pneumoniae)                                                            3      β-N-acetyl-D-                                                                       2.5 units in 0.1M                                                                         N-acetyl 386                                           hexosaminidase                                                                           sodium citrate                                                                            glucosamine                                            (jack bean)                                                                              phosphate, pH 4.5                                           4      α-D-mannosidase                                                                    6 units in 0.1M sodium                                                                    mannose  366                                           (jack bean)                                                                              acetate, 0.01M zinc                                                           acetate, pH 5.0                                             5      β-D-mannosidase                                                                     0.3 units in 0.1M                                                                         mannose  171                                           (Helix pomatia)                                                                          sodium acetate, pH 4.0                                      6      β-N-acetyl-D-                                                                       2.5 units in 0.1M                                                                         N-acetyl 163                                           hexosaminidase                                                                           sodium citrate                                                                            glucosamine                                            (jack bean)                                                                              phosphate, pH 4.5                                           7      α-L-fucosidase                                                                     20 milliunits in 20 mM                                                                    fucose   174                                           (bovine epididymis)                                                                      sodium citrate/                                                               phosphate pH 6.0                                            __________________________________________________________________________

The order and ratio of monosaccharides release was as follows:

D-galactose β1→4: 2 residues

N-acetyl-D-hexosamine β1→2 (Man α1 residue

N-acetyl-D-hexosamine β1→2 (Man α1→6): 1 residue

N-acetyl-D-hexosamine β1→4 (Man β1→4): 1 residue

D-mannose α1→6,3: 2 residues

D-mannose β1→4: 1 residue

L-fucose α1→6: 1 residue

N-acetyl-D-hexosamine β1→4: 1 residue

From this information the sequence of the initial oligosaccharide canclearly only be that as shown in FIG. 42 of the accompanying drawings.

EXAMPLE 3

In this Example an oligosaccharide entity comprising an oligosaccharideof the structure given in FIG. 43 of the accompanying drawings was usedto demonstrate use of the present invention.

A preliminary analysis of the oligosaccharide was carried out and thefollowing monosaccharide units were identified: Man (3 units), Glcnac (3units).

The oligosaccharide (0.3 mg) was attached to a support material asdisclosed in relation to Example 1 to give a conjugated material.

The results of the preliminary analysis were used, in conjunction with ameans for selecting a sequencing agent to be applied to anoligosaccharide entity (said means including a logic unit), to identifycandidate structures and to select a sequencing agent to be applied tothe conjugated material.

The conjugated material was subjected to successive treatments withvarious sequencing agents (in this Example exoglycosidase enzymes), thechoice of each successive sequencing agent being based upon the resultsof analysis and use of a means for selecting a sequencing agent to beapplied to an oligosaccharide entity (said means including a logicunit); the procedures used in this Example were substantially similar tothose disclosed in relation to Example 1.

The order in which the various sequencing agents (exoglycosidases inthis Example) were applied and the results are set out in Table 3.

The order and ratio of monosaccharides release was as follows:

D-mannose α1→3: 1 residue

N-acetyl-D-glucosamine β1→2: 1 residue

D-mannose α1→6: 1 residue

D-mannose β1→4: 1 residue

N-acetyl-D-glucosamine β1→4: 1 residue

From this information the sequence of the initial oligosaccharide canclearly only be that as shown in FIG. 43 of the accompanying drawings.

EXAMPLE 4

In this Example an oligosaccharide entity comprising an oligosaccharideof the structure given in FIG. 44 of the accompanying drawings was usedto demonstrate the use of the present invention.

                                      TABLE 3                                     __________________________________________________________________________    Order of Use      Exoglycosidase Reaction                                                                            Nanomoles of                           of Exoglyco-      Condition (all in 100 μl                                                               Monosaccharide                                                                         Monosaccharide                         sidase Exoglycosidase Used                                                                      reaction volume)                                                                          Detected Detected                               __________________________________________________________________________    1      α-D-mannosidase                                                                    1.4 units in 0.1M                                                                         mannose  157                                           (jack bean)                                                                              sodium acetate/0.01M                                                          zinc acetate, pH 5.0                                        2      β-N-acetyl-D-                                                                       48 μunits in 0.1M                                                                      N-acetyl 141                                           hexosaminidase                                                                           sodium cacodylate, pH                                                                     glucosamine                                            (Streptococcus                                                                           6.0                                                                pneumoniae)                                                            3      α-D-mannosidase                                                                    6 units in 0.1M sodium                                                                    mannose  145                                           (jack bean)                                                                              acetate/0.01M zinc                                                            acetate, pH 5.0                                             4      β-D-mannosidase                                                                     0.3 units in 0.1M                                                                         mannose  151                                           (Helix pomatia)                                                                          sodium acetate, pH 4.0                                      5      β-N-acetyl-D-                                                                       2.5 units in 0.1M                                                                         N-acetyl 142                                           hexosaminidase                                                                           sodium citrate                                                                            glucosamine                                            (jack bean)                                                                              phosphate, pH 4.5                                           __________________________________________________________________________

A preliminary analysis of the oligosaccharide was carried out and thefollowing monosaccharide units were identified: NANA (sialic acid) (2units), Gal (2 units), Glcnac (4 units), Man (3 units).

The oligosaccharide (0.4 mg) was attached to a support material asdisclosed in relation to Example 1 to give a conjugated material.

The results of the preliminary analysis were used, in conjunction with ameans for selecting a sequencing agent to be applied to anoligosaccharide entity (said means including a logic unit), to identifycandidate structures and to select a sequencing agent to be applied tothe conjugated material.

The conjugated material was subjected to successive treatments withvarious sequencing agents (in this Example exoglycosidase enzymes), thechoice of each successive sequencing agent being based upon the resultsof analysis and use of a means for selecting a sequencing agent to beapplied to an oligosaccharide entity (said means including a logicunit); the procedures used in this Example were substantially similar tothose disclosed in relation to Example 1.

The order in which the various sequencing agents (exoglycosidases inthis Example) were applied and the results are set out in Table 4.

                                      TABLE 4                                     __________________________________________________________________________    Order of Use      Exoglycosidase Reaction                                                                            Nanomoles of                           of Exoglyco-      Condition (all in 100 μl                                                               Monosaccharide                                                                         Monosaccharide                         sidase Exoglycosidase Used                                                                      reaction volume)                                                                          Detected Detected                               __________________________________________________________________________    1      α-D-neuraminidase                                                                  0.1 unit in 0.1M sodium                                                                   NANA     194                                           (Arthrobacter                                                                            acetate, pH 5.0                                                    ureafaciens)                                                           2      β-D-galactosidase                                                                   1 unit in 0.1M sodium                                                                     Galactose                                                                              176                                           (jack bean)                                                                              citrate/phosphate,                                                            pH 3.5                                                      3      β-N-acetyl-D-                                                                       48 μunits in 0.1M                                                                      N-acetyl 187                                           hexosaminidase                                                                           sodium cacodylate,                                                                        glucosamine                                            (Streptococcus                                                                           pH 6.0                                                             pneumoniae)                                                            4      α-D-mannosidase                                                                    6 units in 0.1M sodium                                                                    mannose  201                                           (jack bean)                                                                              acetate/0.01M zinc                                                            acetate, pH 5.0                                             5      β-D-mannosidase                                                                     0.3 units in 0.1M                                                                         mannose   90                                           (Helix pomatia)                                                                          sodium acetate,                                                               pH 4.0                                                      6      β-N-acetyl-D-                                                                       2.5 units in 0.1M                                                                         N-acetyl  83                                           hexosaminidase                                                                           sodium citrate                                                                            glucosamine                                            (jack bean)                                                                              phosphate, pH 4.5                                           __________________________________________________________________________

The order and ratio of monosaccharides released was as follows:

D-NANA α2→6: 2 residues

D-galactose β1→4: 2 residues

N-acetyl-D-glucosamine β1→2: 2 residues

Mannose α1→6,3: 2 residues

Mannose β1→4: 1 residue

N-acetyl-D-glucosamine β1→4: 1 residue

From this information the sequence of the initial oligosaccharide canclearly only be that as shown in FIG. 44 of the accompanying drawings.

We claim:
 1. A process for sequencing a primary oligosaccharide compoundwhich is an oligosaccharide or has an oligosaccharide portion, whichcomprises:(a) making a first analysis to determine a monosaccharidecomposition of said primary oligosaccharide compound; and (b) selectingat least one first sequencing agent based on said first analysis; andthen (c) applying said at least one first selected sequencing agent tosaid primary oligosaccharide compound, to give products comprising aresidual oligosaccharide compound and at least one monosaccharidereleased by said at least one first selected sequencing agent; (d)making a second analysis of said at least one monosaccharide in relationto said monosaccharide composition and thereby selecting, based on saidsecond analysis, at least one second sequencing agent for reaction withsaid residual oligosaccharide compound.
 2. A process as claimed in claim1, comprising repeating steps (c) and (d) until a desired degree ofsequencing of said primary oligosaccharide compound has occurred.
 3. Aprocess as claimed in claim 1, wherein the process includes the use of asupport material upon which said primary oligosaccharide compound to besubjected to sequencing is immobilized.
 4. A process as claimed in claim3 wherein the support material is a solid support material comprising1,1' carbonyldiimidazole activated agarose.
 5. A process as claimed inclaim 1, wherein said primary oligosaccharide compound is in freesolution.
 6. A process as claimed in claim 1, wherein at least one saidselected sequencing agent is a chemical agent.
 7. A process as claimedin claim 6, wherein said chemical sequencing agent is an enzymecomprising an exoglycosidase or an endoglycosidase.
 8. A process asclaimed in claim 7 wherein the enzyme is achatina fulica betamannosidase, a.saitoi alpha mannosidase, jack bean alpha mannosidase,bovine testis beta galactosidase, jack bean beta galactosidase, c.lampasbeta xylosidase, s.pneum beta N-acetyl hexosaminidase, jack bean betan-acetyl hexosaminidase, bovine epididymis alpha fucosidase, c.lampasalpha fucosidase, coffee bean alpha galactosidase, or almond alphafucosidase.
 9. A process as claimed in claim 1, wherein at least one ofthe at least one first sequencing agent and the at least one secondsequencing agent comprises a combination of sequencing agents.
 10. Aprocess as claimed in claim 1, further comprising after step (a),preparing a postulated set of possible structures, the postulated setbeing selected from a set of oligosaccharides having monosaccharides inan amount corresponding to the monosaccharide composition of saidprimary oligosaccharide compound based on said first analysis.
 11. Aprocess as claimed in claim 10, wherein step (b) is further based onsaid postulated set of possible structures.